STAPHYLOCOCCI, STREPTOCOCCI PRACTICAL PART 1. IDENTIFICATION OF STAPHYLOCOCCI Free plasmacoagulase • Principle: plasmacoagulase enzyme converts plasma soluble fibrinogen into insoluble fibrin • Tools: test-tube with 0.5 mL of rabbit plasma, fresh staphylococcal culture, loop • Procedure: Inoculate several staphylococcal colonies into the test tube with plasma, incubate 24 hours at 37°C • Evaluation: Observe clot (coagulation) formation after 1 and 24 hours Bound coagulase • Principle: bound coagulase (Clumping factor) is part of the surface of coagulase-positive staphylococci – It leads to clumping of staphylococci and their protection against phagocytosis • Tools: staphylococcal culture, plasma, microscopic slide, loops • Procedure: drop rabbit plasma on the slide, add staphylococcal culture. Mix it properly. • Evaluation: observe agglutination in case of S. aureus Catalase test • Principle: using for differentiation between staphylococci from streptococci • Staphylococci produce catalase enzyme (disintegration of hydrogen peroxide – bubbles formation). Typical feature of aerobic or facultative anaerobic bacteria. • Tools: culture of staphylococci and streptococci, hydrogen peroxide, microscopic slide, loops, disinfection • Procedure: Add few collonies of bacterial culture on the slide than drop hydrogen peroxide onto culture. Observe bubbles formation. • Evaluation: observe positivity (bubbles formation) in staphylococci, negativity in streptococci PRACTICAL PART 2. IDENTIFICATION OF STREPTOCOCCI Optochin test • Principle: Streptococcus pneumoniae is differentiated from other viridans streptococci on the basis of its susceptibility to antibiotic - optochin (Ethyl Hydrocuprein) Optochin test – practice • Tools: Streptococcus pneumoniae and viridans streptococci cultures, blood agar, optochin discs, loops, markers, disinfection • Procedure: inoculate several colonies of Str. pneumoniae on the first half of blood agar, viridans streptococci on the second half. Cultures of both strains must not overlapped!!! • Put optochin disc on each strains and incubate 18-24 hours at 37°C, in 5% CO2 • Evaluation: measure the zone of inhibition, in case of more than 14 mm report as susceptible PRACTICAL PART 3. IDENTIFICATION OF BETA-HEMOLYTIC STREPTOCOCCI – CAMP TEST CAMP test • Principle: group B streptococci (Streptococcus agalactiae) produce extracellular protein (CAMP factor), which is able potentiate beta- hemolysin of Staphylococcus aureus CAMP test – practice • Tools: bacterial culture of tested strain, blood agar, St. aureus strain, marker, loops, disinfection • Procedure: inoculate the line of tested streptococcal strain on blood agar and perpendicularly to this line inoculate the line of St. aureus • Incubation 24 hours at 37°C, aerobically PRACTICAL PART 4. DIFFERENTIATION OF ENTEROCOCCI FROM STREPTOCOCCI Bile - esculin test Principle: •Differentiation of enterococci from other streptococci using cultivation in highly selective- diagnostic bile – esculin medium •Bile inhibits the growth of streptococci •Enterococci hydrolyzes esculin and medium color turns black Bile - esculin test Tools: bacterial culture of tested strain, bile-eskulin medium, marker, loops Procedure: • Pick tested bacterial culture using the loop and inoculate via performing the puncture through the medium to the bottom of tube. • Incubation 24 hours at 37°C, aerobically Evaluation: Positive reaction – blackening of medium Negative reaction – color of medium stays unchanged PRACTICAL PART 5. IDENTIFICATION OF STREPTOCOCCI – EVALUATION CAMP test • Evaluation: positive test is shown as enhancement of beta-hemolysis of tested strain (butterfly wings like) in case of group B streptococci Optochin test • Evaluation: Measure the zones of inhibition around antibiotics after the incubation. Notice the diameter and interpret result of identification according to optochin susceptibility Inhibition zone ≥ 14 mm Report as susceptible Streptococcus pneumoniae Inhibition zone < 14 mm Report as resistant viridans streptococci PRACTICAL PART 6. IDENTIFICATION OF VIRIDANS STREPTOCOCCI – BILE SOLUBILITY TEST Confirmation of identification of Streptococcus pneumoniae – bile solubility test Principle: • Deoxycholate (bile) activates autolysin in cell wall of Str. pneumoniae • Str. pneumoniae colonies are dissolved in presence of deoxycholate • Difference – viridans streptococci colonies show no change in presence of deoxycholate (absence of autolysin in cell wall) Bile solubility test - practice • Tools: Str. pneumoniae and viridans streptococci colonies, 10% solution of sodium deoxycholate • Procedure: Drop deoxycholate on tested colony (colonies). After 15 min observe and report if colony was dissolved or not. • Evaluation: Dissolved colonies – report as Str. pneumoniae Colonies are intact – viridans streptococci PRACTICAL PART 7. LATEX AGGLUTINATION OF BETA- HEMOLYTIC STREPTOCOCCI Detection of group antigen of beta- hemolytic streptococci Principle: Typing of beta-hemolytic streptococci according to specific type of polysaccharide C in streptococcal cell wall (Lancefield antigen). Commercial sets – known types of antibodies (against different types of Lancefield antigens) bound on latex particles. Antigen presence will appear as agglutination with specific antibody Tools: Group specific antisera (antibodies on latex particles), cell wall extract of streptococcal strain, plates, droppers, sticks, disinfection Latex agglutination of beta-hemolytic streptococci • Procedure: transfer one drop of extract (prepared from 5 colonies of tested strain in advance) to each of the five black circles labeled A, B, C, F, G. • Shake the bottles with latex particles anti-A, B, C, F and G and add on drop close to the drop of extract according to the label. Mix the contents in each circle in turn with a stick and spread to cover area of the circle • Rock the card gently for a maximum of 1 minute and read the reaction. Latex agglutination of streptococci Evaluation: •Observe the formation of agglutination during the rotation •http://www.pro-lab.com/products-strep.php?country=DK •https://www.youtube.com/watch?v=xicBT3FaSM0 PRACTICAL PART 8. EVALUATION OF BILE-ESCULIN TEST Bile - esculin test After 24 hours incubation at 37°C, aerobically evaluate the colour change o medium Evaluation: Positive reaction – blackening of medium Negative reaction – color of medium stays unchanged .